Imunocapture Magnetic Beads Enhanced and Ultrasensitive CRISPR-Cas13a-Assisted Electrochemical Biosensor for Rapid Detection of SARS-CoV-2

Abstract

The rapid and ongoing spread of the coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emphasizes the urgent need for an easy and sensitive virus detection method. Here, we describe an immunocapture magnetic bead-enhanced electrochemical biosensor for ultrasensitive SARS-CoV-2 detection based on clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, collectively known as CRISPR-Cas13a technology. At the core of the detection process, low-cast and immobilization-free commercial screen-printed carbon electrodes are used to measure the electrochemical signal, while streptavidin-coated immunocapture magnetic beads are used to reduce the background noise signal and enhance detection ability by separating the excessive report RNA, and a combination of isothermal amplification methods in the CRISPR-Cas13a system is used for nucleic acid detection. The results showed that the sensitivity of the biosensor increased by two orders of magnitude when the magnetic beads were used. The proposed biosensor required approximately 1 h of overall processing time and demonstrated an ultrasensitive ability to detect SARS-CoV-2, which could be as low as 1.66 aM. Furthermore, owing to the programmability of the CRISPR-Cas13a system, the biosensor can be flexibly applied to other viruses, providing a new approach for powerful clinical diagnostics.

Keywords: CRISPR Cas13a; bioanalytical chemistry; biosensor; electrochemistry; trans-acting cleavage.

Figure 1

Principle of the electrochemical Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) biosensor for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. (a) SARS-CoV-2 RNA is extracted from a sample, reverse transcription recombinase-aided amplification (RT-RAA)-amplified, T7 transcribed, and then subjected to the LwaCas13a (Cas13a) trans-cleavage reaction. The Cas13a-crRNA duplex binds target SARS-CoV-2 RNA (140 nt), which activates the trans-cleavage activity. The reRNA, modified with biotin and methylene blue (MB), is then cleaved, and the unbound MB-oligonucleotide is used in the electrochemical measurement. (b) The main process of the electrochemical CRISPR biosensor. When the target RNA is present, the function of Cas13a-crRNA is activated, and the reRNA is cleaved into MB-oligonucleotide and biotin-oligonucleotide. The extra uncleaved reRNA and biotin-oligonucleotide are removed by SA-coated magnetic beads, leaving the MB-oligonucleotide in the solution, which could be detected by electrochemical measurement. When the target RNA is absent, the function of Cas13a-crRNA is not activated. No cleavage of reRNA occurs in the solution, and the reRNA is removed by streptavidin (SA)-coated magnetic beads. Therefore, almost no electrochemical signal can be observed.

Figure 2

Feasibility analysis of the electrochemical CRISPR biosensor. (a) Feasibility analysis of the separation/removal of reRNA using SA-coated magnetic beads. (b) Real-time fluorescence analysis of the feasibility of the Cas13a system. (c) Example electrochemical signal and (d) Square wave voltammetry (SWV) analysis of the feasibility of the Cas13a system for reRNA cleavage. Values represent means ± Standard Deviation (SD), where n = 5 replicates. An unpaired two-tailed Welch’s t-test was used to analyze statistical significance (*** p < 0.001, **** p < 0.0001).

Figure 3

Performance evaluation of the electrochemical CRISPR biosensor without target RNA amplification. (a) SWV signals to various concentrations of target RNA from 0.1 pM to 10,000 pM. (b) Sensitivity analysis, corresponding to (a). (c) Specificity analysis between wild-type strain between two different SARS-CoV-2 variant sites strain(HV69-70del and D614G). The red horizontal dashed line (b) indicates the cutoff current value defined by the average value of the negative control without target RNA (NC) plus three times the SD. The red-filled dots (b) means the current value was larger than the cutoff current value, while the black-filled dots (b) means the current value was smaller than the cutoff current. Values represent means ± SD, where n = 3 replicates. An unpaired two-tailed Welch’s t-test was used to analyze the statistical significance.

Figure 4

Performance evaluation of the electrochemical CRISPR biosensor with RT-RAA amplification. Sensitivity analysis of (a) the electrochemical CRISPR biosensor with (filled dots) or without (hollow dots) SA-coated magnetic beads enhancement, (b) the RT-QPCR assay, and (c) the fluorescent CRISPR assay were performed with various concentrations of target RNA from 0.166 aM to 1.66 × 10⁴ aM. (d) Specificity analysis with 1.66 × 10⁴ aM RNA of SARS-CoV-2, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hemagglutinin 1 neuraminidase 1 (H1N1), dengue virus (DENV), and Coxiella burnetii (CB). The red horizontal dashed line indicates the cutoff current value of the electrochemical CRISPR biosensor with magnetic beads enhanced (a,d) and the fluorescent CRISPR assay (c). The blue horizontal dashed line (a) indicates the cutoff current value of the electrochemical CRISPR biosensor without magnetic beads enhanced. The red-filled dots (a–d) and hollow blue dots (a) indicate positive results, and the solid black dots (a–d) and black hollow dots (a) indicate negative results in the electrochemical CRISPR biosensor with magnetic beads enhanced assay, electrochemical CRISPR biosensor without SA-coated magnetic beads enhancement, the RT-QPCR assay and the fluorescent CRISPR assay.

Figure 5

Analysis of clinical samples. (a) Electrochemical CRISPR biosensor with RT-RAA amplification test results of seven clinical samples from healthy individuals, NC1 through NC7, are the clinical samples from healthy individuals. (b) Thirty-nine suspected positive clinical samples were also tested using the electrochemical CRISPR biosensor with RT-RAA amplification and real-time fluorescence RT-qPCR (Ct value on each small panel, red and black Ct value represent the sample was judged as positive or negative by the electrochemical CRISPR biosensor with RT-RAA amplification). Sample numbers 1–39 successively represent 39 clinical sample numbers. The horizontal dashed line indicates the threshold (value is 55nA). The curve is the SWV signal of the electrochemical CRISPR biosensor test results. The red curve indicates that the maximum current value exceeds the threshold and is judged as positive. Conversely, the black curve indicates that its maximum current value is less than the threshold value, and the result is judged negative.